Small non-coding RNAs are emerging as key players in virus infections, with numerous roles to promote or restrict viral replication and thereby impac human disease. In contrast to infections with DNA viruses, the interaction landscape between RNA viruses and host cell small RNAs is poorly characterized. This proposal outlines a comprehensive program to analyze the dynamics and function of small RNAs, in particular miRNAs, in the context of RNA virus infections. Our focus is on defining functional interactions between cellular miRNAs and viral RNAs that are important for virus replication (like miR-122 and HCV) or modulation of host cell biology. We have uncovered a sponge effect in which HCV RNA functionally sequesters miR-122, and we hypothesize that this may represent a general mechanism by which RNA viruses can influence host gene expression to their advantage. We have also demonstrated that the miR-122 HCV story is not unique-rather pestiviruses also require interaction with a host miRNA for replication. In contrast to HCV and miR-122 with its two binding sites near the 5' end of genome RNA, bovine viral diarrhea virus requires binding of miR-17 family members at a seed site in the 3'UTR that is completely conserved among pestiviruses. These strong preliminary data have kindled our interest in expanding this search to a broad collection of animal viruses in increasingly biologically relevant systems. Besides continuing mechanistic studies on HCV and the pestiviruses, we will expand this effort to two representative arboviruses of medical importance-West Nile virus and chikungunya virus. These will be studied in mosquito and vertebrate cell cultures but also in mosquito vectors and vertebrate models. To encompass more virulent pathogens, we have established a collaboration with the CDC in Atlanta to examine a number of high containment viruses, including, among others, the filoviruses Ebola and Marburg viruses, and tick-borne flaviviruses. This exploration holds the promise of uncovering novel host-virus interactions required for entire virus genera (like miR-122 for the hepaciviruses), insights that could provide new approaches for treatment and prevention of important human viral infections.